UNIVERSITI PUTRA MALAYSIA
CARBON FLUX ANALYSIS OF LIPID BIOSYNTHESIS PATHWAYS IN OIL PALM (ELAEIS GUINEENSIS JACQ. TENERA)
EMILY QUEK MING POH
FPSK (M) 2002 3
CARBON FLUX ANALYSIS OF LIPID BIOSYNTHESIS PATHWAYS IN OIL PALM (ELAEIS GUINEENSIS JACQ. TENERA)
By
ElVIIL Y QUEK MlNG POH
Thesis Submitted to the School of Graduate Studies, Universiti Putra Malaysia , in Fulfilment of the Requirement for the Degree of Master of Science
Ma"ch 2002
DEDICATION
I dedicate this thesis to my parents and my family.
11
Abstract of thesis presented to the Senate of Universiti Putra Malaysia in fulfilment of the requirement for the degree of Master of Science
CARBON FLUX ANALYSIS OF LIPID BIOSYNTHESIS PATHWAYS IN OIL PALM (ELAEIS GUINEENSIS JACQ. TENERA)
By
EMILY QUEK MING POH
March 2002
Chairman: Associate Professor Ong King Kok, Ph.D.
Faculty: Medicine and Health Sciences
The aim of this study was to investigate the carbon flux through lipid biosynthesis
pathways in the oil palm (Elaeis guineensis Jacq. tenera) using metabolic control
analysis (MCA). Three types of tissue from oil palm, namely liquid culture, plastid and
mesocarp were used. The results showed mesocarp tissue was the most suitable tissue
for carbon flux analysis because it incorporated the radioactive precursors mainly into
triacylglycerol (TAG). Further analysis on different stages of fruit development was
carried out using mesocarp tissue at 12, 1 5 and 20 weeks after anthesis (JV AA). It was
confirmed that 20-W AA mesocarp tissue was the best stage of fruit development for
metabolic flux studies because it reflected biosynthesis of storage lipid. Three modes of
radiolabel introduction into the oil palm fruits were investigated, namely injecting the
radiolabel into the fruits still attached to the palm, injecting the radiolabel into the loose
fruit and injecting the radio label into incubation mixture containing meso carp tissue
slices. The level of radioactivity in fruits attached to the palm was lower than the other
two modes of radiolabel introduction. Carbon flux of lipid b iosynthesis pathways was
modulated by temperature and the inhibitor 2-bromooctanoate. Radiolabels [ 1_14C]
III
acetate and [U_1-lC] glycerol were used to monitor the carbon flux through the lipid
biosynthesis pathways. Temperature caused a constraint in the distribution of
radioactivity at the level of diacylglycerol acyltransferase (DAGAT). Therefore,
DAGAT may be a regulatory enzyme. 2-Bromooctanoate inhibited the carbon flux of
lipid biosynthesis pathways . The overall results of MCA suggested that the control of
carbon flux in the oil palm may be distributed over two blocks of the lipid metabolic
pathway, namely the fatty acid biosynthesis block and TAG formation block. Acetyl
CoA carboxylase (ACCase) plays an important role in fatty acid biosynthesis block
while DAGAT plays an important role in the TAG formation block. The molecular
structure of ACCase was investigated using immunoblotting with streptavidin and
screening of ACCase gene in 1 5-W AA oil palm meso carp cDNA library. Immunoblots
with streptavidin showed the presence of large molecular weight (approximately 180
kDa) multifunctional ACCase and smaller molecular weight (approximately 58 kDa)
multisubunit ACCase in oil palm mesocarp. Screening for ACCase gene in 1 5 -W AA
oil palm mesocarp cDNA library showed several strong signals corresponding to the
putative �-carboxyl transferase subunit of ACCase.
IV
Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai memenuhi keperluan untuk ijazah Master Sains
ANALISIS FLUKS KARBON TAP AK JALAN BIOSINTESIS LIPID DI
DALAM POKOK SAWIT (ELAEIS GUINEENSIS JACQ. TENERA)
Oleh
EMIL Y QUEK MING POH
Mac 2002
Pengerusi: Profesor Madya Ong King Kok, Ph.D.
Fakulti: Perubatan dan Sains Kesihatan
Matlamat kajian ini adalah untuk mengkaji fluks karbon melalui tapak jalan biosintesis
lipid di dalam pokok sawit (Elaeis guineensis Jacq. tenera) dengan menggunakan
analisis kawalan metabolik (MeA). Tiga jenis tisu daripada pokok sawit iaitu kultur
cecair, p lastid dan mesokarpa telah digunakan. Hasil menunjukkan bahawa tisu
mesokarpa merupakan tisu yang sangat sesuai untuk analisis fluks karbon kerana ia
menukarkan prekursor radioaktif kebanyakannya ke dalam triasilgliserol (TAG).
Analisis lanjutan ke atas peringkat perkembangan buah yang berlainan telah dilakukan
dengan menggunakan tisu mesokarpa pada 1 2, 1 5 dan 20 minggu selepas
pendebungaan (W AA). Tisu mesokarpa pada 20 W AA telah disahkan sebagai
peringkat perkembangan buah yang terbaik untuk kajian fluks metabolik kerana ia
mengimbas biosintesis penyimpanan lipid. Tiga cara kemasukan penanda radioaktif ke
dalam buah sawit iaitu menyuntik penanda radioaktif ke dalam buah yang masih di
pokok, menyuntik penanda radioaktif ke dalam buah yang dipetik dan menyuntik
penanda radioaktif ke dalam larutan eraman yang mengandungi hirisan tisu mesokarpa
telah dikaj i. Aras aktiviti radioaktif dalam buah yang masih di pokok adalah lebih
v
rendah berbanding dengan dua cara kemasukan penanda radioaktif yang lain. Fluks
karbon dalam tapak jalan biosintesis lipid telah dimodulasikan o leh suhu dan perencat
2-bromooktanoat. Penanda radioaktif [1 _14C] asetat dan [U_14C] gliserol telah
digunakan di dalam kajian ini untuk memantau fluks karbon melalui tapak jalan
biosintesis lipid. Suhu menyebabkan rencatan dalam penyebaran aktiviti radioaktif
pada peringkat diasilgliserol asiltransferase (DAGAT). Oleh itu, DAGAT mungkin
merupakan enzim pengawalatur. 2-Bromooktanoat mereneat fluks karbon dalam tapak
jalan biosintesis lipid. Hasil keseluruhan MCA mencadangkan kawalan fluks karbon
dalam buah sawit disebarkan melalui dua blok dalam tapak jalan metabolik lipid iaitu
biok biosintesis asid Iemak dan biok pembentukan TAG. AsetiI-CoA karboksilase
(ACCase) memainkan peranan penting dalam biok biosintesis asid Iemak manakaia
DAGAT memainkan peranan penting dalam b lok pembentukan TAG. Stuktur
molekular ACCase telah dikaji dengan menggunakan pembiotan Imuno dengan
streptavidin dan penyaringan gen ACCase dalam koleksi eDNA 1 5-W AA mesokarpa
sawit. Pemblotan imuno dengan streptavidin menunjukkan kehadiran protein berberat
molekul besar (kira-kira 1 80 kDa) iaitu ACCase pelbagai-fungsi dan protein berberat
molekul keeil (kira-kira 58 kDa) iaitu ACCase pelbagai-subunit di dalam mesokarpa
sawit. Penyaringan gen ACCase dalam koleksi eDNA 1 5 -WAA mesokarpa sawit telah
menunjukkan beberapa signal yang menyamai subunit p-karboksil transferase ACCase.
VI
ACKNOWLEDGEMENTS
I would like to express my sincere appreciation and million thanks to my supervisors,
Associate Professor Ong King Kok, Professor Khor Hun Teik and Dr Ravigadevi
Sambanthamurthi for their suggestion, advice, support and guidance throughout my
project.
My appreciation and gratitude go to my parents and my family for their constant
support, love and patient throughout my graduate study. My sincere thanks and
gratitude are also extended to all the staff of Metabolics Laboratory especially Ms. Jane
Sonia, Mr. Andy Yip, En. Jamil, En. Rahim, Pn. Jabariah and Pn. Siti Hasnah for their
help towards the success of this project and also to my friends for their support.
Vll
I certify that an Examination Committee met on 6th March 2002 to conduct the final examination of Emily Quek Ming Poh on her Master of Science thesis entitled "Carbon Flux Analysis of Lipid Biosynthesis Pathways in Oil Palm (Elaeis guineensis Jacq. tenera)" in accordance with Universiti Pertanian Malaysia (Higher Degree) Act 1 980 and Universiti Pertanian Malaysia (Higher Degree) Regulations 1 98 1 . The Committee recommends that the candidate be awarded the relevant degree. Members of the Examination Committee are as follows:
Dr Chan Hooi Har, Ph.D. Associate Professor Faculty of Medicine and Heath Sciences, Universiti Putra Malaysia (Chairman)
Ong King Kok, Ph.D. Associate Professor Faculty of Medicine and Heath Sciences, Universiti Putra Malaysia (Member)
Ravigadevi Sambanthamurthi, Ph.D. Advanced B iotechnology and Breeding Centre, Malaysian Palm Oil Board (Member)
Khor Hun Teik, Ph.D. Professor Faculty of Medicine and Heath Sciences, Universiti Putra Malaysia (Member)
... SHAMSHER MOHAMAD RAMADILI, Ph.D. ProfessorlDeputy Dean, School of Graduate Studies Universiti Putra Malaysia
Date: 5 APR 2002 VUl
This thesis submitted to the Senate of Universiti Putra Malaysia has been accepted as fulfilment of the requirement for the degree of Master of Science.
AINI IDERIS, Ph.D. ProfessorlDean, School of Graduate Studies, Universiti Putra Malaysia
Date�f 3 ./1 ,�, 2002
IX
DECLARATION
I hereby declare that the thesis is based on my original work except for quotations and citations which have been duly acknowledged. I also declare that it has not been previously or concurrently submitted for any other degree at UPM or other institutions
Date: 4 April � OOJ,
x
TABLE OF CONTENTS
Page DEDICATION
ABSTRACT
. . 11
III ABSTRAK ACKNOWLEDGEMENTS APPROVAL DECLARATION
V Vll Vlll X XIV XV XVlll
LIST OF TABLES LIST OF FIGURES LIST OF ABBREVIATIONS
CHAPTER
2
3
INTRODUCTION
LITERATURE REVIEW 2 . 1 Oil Palm 2 .2 Palm Oil 2 .3 Plant Lipids
2 . 3 . 1 Classification of Lipids 2 .3 .2 Functions of Lipids
2.4 Fatty Acid Biosynthesis 2.4. 1 Acyl Carrier Protein (ACP) 2 .4 .2 Acetyl-CoA 2.4 .3 Acetyl-CoA Carboxylase (ACCase) 2.4.4 Fatty Acid Synthase (F AS) 2 .4 .5 Desaturase 2.4. 6 Acyl-ACP Thioesterase
2 .5 Triacylglycerol Biosynthesis 2 .6 The Kennedy Pathway 2 .7 Metabolic Control Analysis (MCA) 2.8 Metabolic Engineering
MATERIALS AND METHODS
1
4 4 7 8 8 1 0 1 0 1 3 1 4 1 5 1 6 1 7 1 8 18 1 9 2 1 26
28 3 . 1 Chemicals and Reagents 28 3 .2 Tagging of Oil Palm Fruits 30 3 .3 Experimental Fruits 30 3 .4 Oil Palm Liquid Cultures 30 3 .5 Preparation of Meso carp Tissue S lices 3 1 3 .6 Isolation of Crude Plastids 3 1 3 .7 Bio-Rad Protein Assay 32 3 . 8 Radioactive Incorporation 32
3 . 8 . 1 [lYC] Acetate Incorporation in Liquid Cultures 33 3 . 8 .2 [1_14C] Acetate Incorporation in Plastids 33 3 . 8 . 3 [1_14C] Acetate or [U_14C] Glycerol Incorporation in
Mesocarp Tissue Slices 34
Xl
3 .9 Mode of Radio label Uptake into the Oil Palm Fruits 34 3 . 1 0 Determination of pH Optimum 35 3 . 1 1 Time Course Experiment of Incorporation of Radio label 3 5 3 . 1 2 Optimisation ofMES-NaOH Concentration 36 3 . 1 3 Effect of Temperature 36 3 . 1 4 Inhibition with 2-Bromooctanoate 36
3 . 1 4 . 1 Preparation of2-Bromooctanoate Solution 36 3 . 1 4 .2 Incubation with 2-Bromooctanoate 3 7
3 . 1 5 Analysis of Radioactive Incorporation Products 37 3 . 1 6 Thin Layer Chromatography (TLC) 38
3 . 1 6 . 1 Preparation ofTLC Plates 38 3 . l 6 .2 Application of Samples onto TLC Plate 39 3 . 1 6 .3 Separation of Lipid Classes 39 3 . 1 6 .4 Detection of Lipids 39 3 . 1 6.5 Determination of Radioactivity of Separated Lipids 40
3 . l 7 Separation of Acyl-ACPs and Acyl-CoAs 40 3 . 1 8 Liquid Scintillation Counting 4 1 3 . l9 Extraction of Proteins 4 1 3 .20 Acetyl-CoA Carboxylase (ACCase) Assay 42 3 .2 1 Sodium Dodecyl Sulphate - Polyacrylamide Gel Electrophoresis
(SDS-PAGE) 42 3 .2 1. 1 Preparation of SDS-P AGE Gel 43 3 .2 1 .2 Electrophoresis Conditions for SDS -PAGE 44 3 .2 1 .3 Development of SDS-PAGE Gel 44
3 .22 vVestern Blotting 44 3 . 22 . 1 Preparation for Western Blotting 45 3 . 22 .2 Assembly of the Western Blotting Unit 45
3 .23 Immunoblotting with Streptavidin 46 3 .23 . 1 Preparation of Detection Solution 47 3 .23 .2 Immunob lotting 47
3 .24 Preliminary ACCase Gene Isolation 48 3 .24 . 1 Preparation o f ACCase Probe 48 3 .24 .2 Screening of ACCase Gene in 1 5-W AA Mesocarp cDNA
Library 54 3 .24 . 3 Storage of Transformed Cells 57
3 .25 Statistical Analysis 57
4 RESULTS AND DISCUSSION 58 4 . 1 Conditions for Incorporation of Radiolabel in Crude Plastids 58 4.2 Determination of Suitable Tissue 60 4 .3 Mode of Radiolabel Uptake into the Oil Palm Fruits 66 4.4 Determination of pH Optimum 68 4 .5 Time Course Experiment of Incorporation of Radiolabel 71 4 .6 Optimisation ofivlES-NaOH Concentration 73 4.7 Effect of Temperature on Lipid Biosynthesis in Oil Palm Mesocarp
Tissue 73 4 .7. 1 Effect of Temperature on the Incorporation of Radioactivity
into Total Lipids in Oil Palm Mesocarp Tissue 73
XII
5
4 .7. 2 Effect of Temperature on the Distribution of Radio label into Lipid Classes in Oil Palm Mesocarp Tissue 76
4 .7 .3 Effect of Temperature on Acyl-ACP and Acyl-CoA Pools in Oil Palm Mesocarp Tissue 78
4 . 8 Manipulation of Lipid Biosynthesis in Oil Palm Mesocarp Tissue by the Inhibitor 2-Bromooctanoate 83
4. 8 . 1 Effect of2-Bromooctanoate on the Incorporation of [ 1 -14C] Acetate into Total Lipids in Oil Palm Mesocarp Tissue 83
4. 8 . 2 Effect of 2-Bromooctanoate on the Incorporation of [ 1 _14C] Acetate into Lipid C lasses in Oil Palm Mesocarp Tissue 85
4 . 8 . 3 Effect of2-Bromooctanoate on Acyl-ACP and Acyl-CoA Pools in Oil Palm Mesocarp Tissue 85
4 .8 .4 Effect of2-Bromooctanoate on the Incorporation of [U}4C] Glycerol into Total Lipids in Oil Palm Mesocarp Tissue 87
4. 8 . 5 Effect of2-Bromooctanoate on the Distribution of [U)4C] Glycerol into Lipid Classes in Oil Palm Mesocarp Tissue 87
4 .9 Application of Metabolic Control Analysis (MCA) on the Lipid Biosynthesis Pathway in Oil Palm Mesocarp Tissue 91
4 . 1 0 Acetyl-CoA Carboxylase (ACCase) 97 4. 1 0 . 1 Sodium Dodecyl Sulphate - Polyacrylamide Gel
Electrophoresis (SDS-PAGE) 97 4. 1 0 .2 Immunoblotting with Streptavidin 99 4. 10 . 3 Preliminary ACCase Gene Isolation 1 02
4 . 1 1 Future Studies 104
CONCLUSION 1 05
REFERENCES 1 07
APPENDIX 1 1 1 7
VITAE 1 1 9
Xlll
LIST OF TABLES
Table Page
2. 1 Lipid classification (King, 1996). 9
4 .1 The percentage (%) incorporation of radioactivity into total lipids in the plastid without and with cofactors, ACP, ATP, NADH and NADPH 59
4.2 The percentage (%) incorporation of [1_14C] acetate into total lipids in the plastid, liquid culture and mesocarp tissue system. 60
4.3 The percentage (%) distribution of [1 }4C] acetate into lipid classes in the plastid, liquid culture and mesocarp tissue system. 63
4.4 The percentage (%) distribution of [1_1 4C] acetate into lipid classes in 1 2 -W AA, 16-W AA and 20-W AA oil palm mesocarp tissue. 64
4 .5 The percentage (%) incorporation of radioactivity into total lipids in three different modes of radiolabel uptake into 20-W AA oil palm fruits . 67
4 .6 The percentage (%) distribution of [1_1 4C] acetate into lipid classes in three different modes of radio label uptake into 20-W AA oil palm fruits. 68
4.7 The calculation of elasticity coefficients and flux control coefficients of the lipid biosynthesis pathway of the oil palm mesocarp tissue affected by temperature perturbation (see Appendix 1). 94
XIV
LIST OF FIGURES
Figure
2. 1 Oil palm fruit.
2.2 Fatty acid biosynthesis pathway in plants.
2 .3 The Kennedy pathway in plants.
2 .4 Top-down metabolic control analysis (TDCA).
2 .5 Bottom-up metabolic control analysis (BUCA).
3. 1 A 25-ml conical flask containing the CO2 trap and the radioactive incubation mixture.
Page
5
1 2
19
25
25
33
3.2 Assembly of the Western blotting unit. 46
4. 1 The separation of radio labelled lipids by TLC. 1 : Radiolabelled lipids extracted from mesocarp tissue. 2: Radiolabelled lipids extracted from plastid. 3: Radiolabelled lipids extracted from liquid cultures. 4: Lipid standards (Sigma). 62
4.2 Optimisation of pH for the incorporation of radioactivity. 70
4 .3 The percentage (%) incorporation of radioactivity measured at one-hour intervals . 72
4.4 The percentage (%) incorporation of radioactivity for vanous concentrations ofwIES-NaOH buffer. 74
4 .5 The percentage (%) incorporation of radioactivity into total lipids at various temperatures.
4 .6 The percentage (%) incorporation of radioactivity into lipid classes at various temperatures.
4 .7 The separation of the acyl-ACPs and acyl-CoAs through the SEP-PAK
75
77
C 1 8 column (Waters). SO
4.S Effect of various temperatures on the incorporation of radioactivity into acyl-ACPs in 20-WAA oil palm mesocarp tissue. Sl
4.9 Effect of various temperatures on the incorporation of radioactivity into acyl-CoAs in 20-WAA oil palm mesocarp tissue. 81
xv
4.10 Effect of vanous concentrations of 2-bromooctanoate on the incorporation of [ 1 _14C] acetate into total lipids in 20-W AA oil palm mesocarp tissue.
4. 1 1 Effect of various concentrations of 2-bromooctanoate on the distribution of [ 1 _14C] acetate into lipid classes in 20-WAA oil palm mesocarp tissue.
4. 1 2 Effect of vanous concentrations of 2-bromooctanoate on the incorporation of radioactivity into acyl-ACPs in 20-W AA oil palm meso carp tissue.
4. 1 3 Effect of vanous concentrations o f 2-bromooctanoate on the incorporation of radioactivity into acyl-CoAs in 20-W AA oil palm meso carp tissue.
4 . 14 Effect of vanous concentrations of 2-bromooctanoate on the incorporation of [U_14C] glycerol into total lipids in 20-WAA oil palm mesocarp tissue.
4 . 1 5 Effect of various concentrations of 2-bromooctanoate on distribution of
84
86
88
88
89
[U_14C] glycerol into lipid classes in 20-WAA oil palm mesocarp tissue. 90
4. 1 6 Top-dovvn approach ofMCA on lipid biosynthesis pathway. 93
4. 1 7 (a) SDS -P AGE using standard gel to separate the crude proteins extracted from 1 5-WAA mesocarp. The standard gel consisted of a separating gel ( 1 2%) and a stacking gel (4%). Well 1 : crude proteins extracted from 1 5-W AA mesocarp, and well 2: high range protein molecular weight markers (Bio-Rad). (b) Graph of 10glO molecular weight against distance of migration. 98
4. 1 8 SDS-PAGE using gradient gel to separate the crude proteins extracted from 1 5 -WAA mesocarp. The gradient gel consisted of a separating gel (5-20%) and a stacking gel (4%). Well 1 and 2 : crude proteins extracted from lS-WAA mesocarp, and well 3 : high range biotin-labelled calibration proteins (Boehringer Mannheim). 98
4. 1 9 Immunoblotting of separated proteins with streptavidin. The highlighted bands indicate the biotin-containing proteins . These proteins were detected using chemiluminescence blotting kit (Boehringer Mannheim). Well 1 : high range biotin-labelled calibration proteins (Boehringer Mannheim), and well 2: crude proteins extracted from lS-WAA mesocarp. 1 0 1
XVl
4.20 Gel electrophoresis of restriction enzyme digestion products on 1 .2% agarose. Well 1 : marker 1 00 bp DNA ladder, well 2 : uncut plasmid DNA, well 3 : plasmid DNA digested with Nco I, well 4: uncut plasmid DNA, well 5 : plasmid DNA digested with Hind III and Bg II, well 6 : uncut plasmid DNA, well 7 : plasmid DNA digested with Hind III and B g II, and well 8: marker 1 kbp DNA ladder. 103
4 .21 Screening of ACCase gene in 1 5 -WAA mesocarp cDNA library. Several strong s ignals corresponding to putative �-ct subunit of ACCase were obtained. 1 03
XVll
ACCase
ACP
ADP
A1vlP ANOVA
ATP
BC
BCCP
BCS
BSA
BUCA
C1 2:0
C 1 6 :0
C 1 8 :0
C1 8 : 1
C1 8:2
C1 8 :3
LIST O F ABBREVIATIONS
Acetyl-CoA carboxylase
Acyl carrier protein
Adenosine biphosphate
Adenosine monophosphate
Analysis of variance
Adenosine triphosphate
Biotin carboxylase
Biotin carboxyl carrier protein
Biodegradable counting scintillant
Bovine serum albumin
Bottom-up metabolic control analysis
Lauric acid
Palmitic acid
Stearic acid
Oleic acid
Linoleic acid
Linolenic acid
CI6:0-ACP Palmitoyl-ACP
CI8 :0-ACP Stearoyl-ACP
CI8: l-ACP Oleoyl-ACP
cpm
CPO
CT
DAG
DAGAT
1 6 carbons
1 8 carbons
Curie
Coenzyme A
Carbon dioxide
Counts per min
Crude palm oil
Carboxyl transferase
Diacylglycerol
Diacylglycerol acyltransferase
XVIll
DNA
EDTA
FAS
FFA
HCl
HC03-
HEPES
H2S04
IPTG
KAS
KCl
kDa
KHC03
KOH
LB
LPC
:tvlAG
MgClz
MgS04
l\1nClz
MCA
MES
NaCl
NADH
NADPH
NaHl4C03
NaOH
PAGE
pfu
pH
PKO
Deoxyribonucleic acid
Ethylenediaminetetraacetic acid
Fatty acid synthase
Free fatty acid
Hydrochloric acid
Ion bicarbonate
N-[2-Hydroxylethyl]piperazine-N' -2-ethanesulphonic acid
Sulphuric acid
Isopropylthio-p-D-galactoside
p-Ketoacyl-ACP synthetase
Potassium chloride
Kilo Dalton
Potassium bicarbonate
Potassium hydroxide
Luria-Bertani
Lysophosphatidylcholine
Monoacylglycerol
Magnesium chloride
Magnesium sulphate
Manganous chloride
Metabolic control analysis
2-[N-morpholino ]ethanesulphonic acid
Sodium chloride
Nicotinamide adenine dinucleotide reduced form
Nicotinamide adenine dinucleotide phosphate reduced fonn
Sodium e4C] bicarbonate
Sodium hydroxide
Polyacrylamide gel electrophoresis
Plaque fonning unit
Hydrogen potential
Palm kernel oil
XIX
PL
POD
PI
PPI
ppm
PVDF
SD
SDS
sn SOC
SPSS
SSC
TAG
TBS
TBST
TCA
TDCA
TLC
V
var.
v/v
WAA
w/v
X-gal
Phospholipid
Peroxidase
Inorganic phosphate
Pyrophosphate
Part per million
Polyvinylidene difluoride
Standard deviation
Sodium dodecyl sulphate
Stereospecific number
Sodium citrate
Statistical package for social sciences
Sodium-citrate saline
Triacylglycerol
Tris-buffered saline
TBS-Tween 20
Tricarboxylic acid
Top-down metabolic control analysis
Thin layer chromatography
Volt
Variety
Volume/volume
Weeks after anthesis
Weight/volume
5-Bromo-4-chloro-3 -indolyl-� -D-galactoside
xx
CHAPTER 1
INTRODUCTION
Recent advances in biotechnology, such as in genomics, proteomics, DNA microarray
and bioinformatics, have enabled plants to be modified to produce novel products.
These novel products include biodegradable plastics (Poirier et aI. , 1 992), antibodies
(Hiatt et aI. , 1 989; Ma and Hein, 1 995) and interferon (De Zoeten et aI., 1 989).
Palm oil has become an important edible oil over the last few decades with a
production of about 1 8 .2 million tonnes in 1 996-2000 from 3 .7 million tonnes in 1 976-
1 980 when it supplied a mere 7 . 1 % of the world's oils and fats (Basiron, 2000). It now
contributes 23 % to the world's oils and fats production, making it the second-most
produced oil after soybean oil (Gunstone, 200 1 ). The oil palm is the highest-yielding
oil crop in the world, surpassing the coconut (the next highest-yielding o il crop) by
about 50-1 00% and other oil crops by even more (Basiron, 2000).
Malaysia is the largest producer of palm oil with 60% of the world production (Chow,
1 997). Palm oil is predicted to become the major oil in the world by 201 2 (Oil World,
1 999), but the stiff competition from other oils has made it necessary to diversify its
use. In addition, novel higher value-added products can be made producible by the oil
palm by employing biotechnology methods such as recombinant DNA technology and
genomics (Cheah, 1 999). However, this requires a detailed understanding of the basic
plant metabolism.
Plant lipid biosynthesis has been much studied in recent years (Browse and SomelVille,
1 99 1 ; Harwood, 1 999 ; Ohlrogge and Jaworski, 1 997; Slabas and Fawcett, 1 992) but
there remains much more to be learnt. To manipulate the oil palm for novel oils such as
high oleate (Cheah, 1 997) would require substantially more information on the
regulation of lipid biosynthesis.
As metabolic pathways are under multi-step control, unraveling a single step or an
individual enzyme is insufficient to understand the entire metabolism. Indeed, the
converse is needed - to have an overall picture of the metabolic pathway before the
particular steps can be understood. This can be achieved by applying the metabolic
control analysis (MCA).
In most plants , including the oil palm, the storage lipids are mainly triacylglycerols
(TAGs) (Harwood, 1980; Murphy, 1 993). The metabolic pathways to TAG involve
acetyl-CoA as the immediate carbon source, and information on the metabolic flux will
be useful in modeling the carbon flux through pathways . This work was therefore to
investigate the carbon flux in lipid biosynthesis by the oil palm using a radiolabel. It is
hoped that the information gained may be useful in diverting the carbon to the
formation of higher-value products by genetic manipulation.
To increase the production of a metabolite(s), it is necessary to manipulate a reaction,
or a set of reactions, over another. However, the manipulation may still not result in
production of the metabolite(s) if the necessary control mechanisms are not in place.
Therefore, a comprehensive understanding of the entire cellular metabolism is needed.
2
Currently, attempts are being made to design cellular metabolism in order to maximize
output of the desired metabolites . But the requisite prelude to this is quantification of
the metabolite flux by MCA.
The objectives of this research were to :
1 ) Investigate carbon flux through the oil palm lipid biosynthesis pathway(s).
2) Apply MCA for a better understanding of the overall quantitative control structure
of the lipid biosynthesis pathway(s).
3
CHAPTER 2
LITERATURE REVIEW
2.1 Oil Palm
Oil palm is a perennial oil-bearing crop that has an economic life of about 25 years
(Gascon et aI., 1 989; Basiron, 200 1 ). It begins to bear fruit two to three years after
planting (Basiron, 200 1 ). It is a unique crop that yields two types of oil, crude palm oil
(CPO) from the mesocarp of the fruit and palm kernel oil (PKO) from the seed or
kernel. These two oils have different physical and chemical properties. CPO contains
mainly palmitic acid (C1 6 :0) and oleic acid (C1 8 : 1 ), the two most common fatty acids
in nature while PKO contains mainly lauric acid (C1 2 :0).
The oil palm fruit is a sessible drupe which is usually oval in shape. The length of the
fruit is 2 .5 to 5.0 cm and 2 .5 cm in diameter. It may weigh from 3 to 30 g (Godin and
Spensley, 1 971 ; Gascon et aI., 1 989).
The oil palm fruit consists of the mesocarp, the shell or endocarp and the kernel as
shown in Figure 2 . 1 . The mesocarp or pulp of the ripe oil palm fruit is yellowish
orange in colour. It is oily and fibrous (Vaughan, 1 970). The outer layer of oil palm is
called exocarp or epicarp. It shows variation in colour through yellow, orange, red,
brown and black according to the variety (Cobley and Steele, 1 976).
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